Do you sonicate before adding to GST beads (slurry)? - (Feb/07/2006 )

molecular cloning says dont sonicate....but some papers use sonication....what do you usually do? thanx a lot

-Kathy-

QUOTE (Kathy @ Feb 8 2006, 08:01 AM)

molecular cloning says dont sonicate....but some papers use sonication....what do you usually do? thanx a lot

You should sonicate to break the bacteria and centrifuge to separate the membranes from the cytosol if your protein is soluble. You could avoid sonication if you use lysozime in the buffer which breaks the cellular wall by itself.

-Nagroc-

it says that after lysozyme I add 0.2% triton x-100, shake tube vigorously add RNase and DNase then cetrifuge and remove debris (cell walls etc.??) does it seem ok or it is better to sonicate? thanx again

-Kathy-

I have tried to use lysozyme or several freeze-thaw cycles to break the bacterial cells without sonication. Both are fine. As you said, lysozyme and 0.2% trixon x-100 are added, sonication is not need.

-Patrickn-

QUOTE (Kathy @ Feb 9 2006, 04:23 AM)

it says that after lysozyme I add 0.2% triton x-100, shake tube vigorously add RNase and DNase then cetrifuge and remove debris (cell walls etc.??) does it seem ok or it is better to sonicate? thanx again

If you make a treatment with lysozyme, as you said, definately sonication is no needed.

-Nagroc-

i'm ok! if you treat the cell with lysosyme , you don't have to sonicate, Triton X100 at 0.2 to 1% may help to solubilize your protein, moreover for a 50 ml cell pellet , you can process a 6 cycle sonication step= 30'' sonication on ice/30'' on ice . i have tried this protocle and i have a better yield extraction, if you are not sure that your protein is soluble, try to check the insoluble fraction by loading it on an SDS-PAGE gel in denatured condition